Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period.2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms).3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change.4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation.5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics.6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity.7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant.8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

Stimulation of microsecond-delayed fluorescence from spinach chloroplasts by uncouplers and by phosphorylation

Delayed fluorescence, as measured with a laser phosphoroscope, is stimulated not inhibited by uncouplers during the first 100 μs after the light is turned off. This is true only wen uncouplers cause an increase in the rate of electron transport. When ADP and P_i cause an increase in the electron transport rate, microsecond-delayed fluorescence is also increased. Indeed, there is a complex quantitative relationship between the rate of electron transport and the initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P_i also increase the rate of decay of delayed fluorescence so that after about 150 μs they become inhibitory, as already reported by many authors.

Microsecond-delayed fluorescence continues to rise with rising light intensities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport both with the intensity of the 5–100 μs-delayed fluorescence and with the rate of decay in the intensity of delayed fluorescence. The data imply that the decrease in intensity of millisecond-delayed fluorescence which has often been noted with uncouplers is probably not due to the elimination of a membrane potential. It seems more likely that the decrease in millisecond-delayed fluorescence is a reflection of the rate of disappearance of some other electron transport-generated condition, a condition which is uncoupler-insensitive. Certainly stimulations of microsecond-delayed fluorescence by electron transport which has been uncoupled by gramicidin suggest that ion gradients are not an essential component of the conditions responsible for delayed fluorescence.     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.     

Changes in the redox state of the secondary acceptor of Photosystem II associated with light-induced thylakoid protein phosphorylation

Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the $\text{B}{}^{\mathrm{?}}\text{B}$ ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B^?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.     

Regulation of photosystem stoichiometry,chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure

The structural-functional organization of higher plant chloroplasts has been investigated in relation to the particular light conditions during plant growth. (1) Light intensity variations during growth caused changes in the $\text{Chl}\text{a}\text{Chl}\text{b}$ ratio, in the light-saturated uncoupled rates of electron transport to a Hill oxidant and in the distribution of the chloroplast volume between the membrane and stroma phases. (2) Light quality differences during growth had an effect on the PS II/PS I reaction center ratio and on the chloroplast membrane phase differentiation into grana and stroma thylakoids. Plants grown under far-red-enriched (680–710 nm) illumination contained higher (20–25%) amounts of PS II and simultaneously lower (20–25%) amounts of PS I reaction centers. They also showed a higher grana density along with thicker grana stacks in their chloroplasts. (3) The size of the light-harvesting antenna pool of PS II centers was estimated from the fluorescence time course of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts and was found to be fairly constant (±10%) in spite of the variable PS II/PS I reaction center ratio. The results are compatible with the hypothesis that the structural entities of grana facilitated the centralization and relative concentration increase of a certain group of PS II reaction centers.     

Isolation and characterization of Photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N₂ fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome $\text{b-559}$ ratio of 50 : 1. The Photosystem I particle is highly enriched in $\text{P-700}$, with a chlorophyll to $\text{P-700}$ ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

Photoactivation of the manganese catalyst of O2 evolution. I. Biochemical and kinetic aspects

Photosynthetic O₂ evolution requires a Mn complex which is activated by light. An analysis of this activation process yielded the following results:

1.

1. In any given illumination, the time course is first order, the rate being proportional to the number of inactive O₂-evolving System II trapping centers (the quantum yield being invariant).     

Bicarbonate effects on chlorophyll a fluorescence transients in the presence and the absence of diuron

We investigated the effect of HCO^?₃ addition to CO₂-depleted thylakoids by means of fluorescence techniques. (1) In the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), the net reduction of the primary quinone-type electron acceptor (Q) of Photosystem (PS) II is about 2-times faster in the absence of HCO^?₃ than in its presence, whether normal, heat-treated or NH₂OH-treated samples are used. This effect of HCO^?₃ is, therefore, not on the O₂-evolving apparatus. It is, however, interpreted to be due to an influence of HCO^?₃ on the kinetics of the reduction of Q, perhaps combined with an effect on the back reaction of Q^? with P-680⁺, the oxidized form of the PS II reaction center chlorophyll a. (2) Fluorescence experiments in the absence of diuron indicate that the absence of HCO^?₃ results in a complete block at the quinone level; the area over the fluorescence induction curve in the absence of HCO^?₃ was found to be 2.2-times higher in the absence than in the presence of diuron, pointing to a complete block of BH₂ oxidation in the absence of HCO^?₃. (3) No change in the midpoint potential of Q is observed when HCO^?₃ is added to CO₂-depleted membranes. HCO^?₃ not only has a large (on/off) effect on the reoxidation of BH₂, but also a smaller effect between P-680 and Q. We propose that HCO^?₃ binding to its specific site in the thylakoid membrane results in a conformational change, allowing normal electron transport between the two photosystems.     

A major site of bicarbonate effect in system II reaction. Evidence from ESR signal IIvf, fast fluorescence yield changes and delayed light emission

In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal II_vf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680⁺; (4) electron flow from Q^? to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.